Single-molecule techniques offer a unique tool for studying the dynamical behavior of individual molecules and provide the possibility to construct distributions from individual events rather than from a signal stemming from an ensemble of molecules. In biological systems, known for their complexity, these techniques make it possible to gain insights into the detailed spectrum of molecular conformational changes and activities. Here, we report on the direct observation of a single lipase-catalyzed reaction for extended periods of time (hours), by using confocal fluorescence microscopy. When adding a profluorescent substrate, the monitored enzymatic activity appears as a trajectory of “on-state” and “off-state” events. The waiting time probability density function of the off state and the state-correlation function fit stretched exponentials, independent of the substrate concentration in a certain range. The data analysis unravels oscillations in the logarithmic derivative of the off-state waiting time probability density function and correlations between off-state events. These findings imply that the fluctuating enzyme model, which involves a spectrum of enzymatic conformations that interconvert on the time scale of the catalytic activity, best describes the observed enzymatic activity. Based on this model, values for the coupling and reaction rates are extracted.

The combination of polymeric with biological materials, to create biohybrid macromolecules that merge the properties of both the natural and synthetic components, is a flourishing area in both life sciences and biotechnology. The click chemistry philosophy has recently provided a powerful tool in this direction, leading to a plethora of novel, tailor‐made biomacromolecules with unprecedented structural characteristics and properties. The different synthetic strategies, using the alkyne–azide click cycloadditions to bioorthogonally achieve the coupling of synthetic polymers with nucleic acids, peptides, sugars, proteins or even viruses and cells is described. The review covers the latest developments in this very dynamic and rapidly expanding field. magnified image